Type 1 diabetes (T1D) develops as a result of insufficient insulin being produced due to a self-destructive immune response against insulin producing beta cells. Although a number of factors are known to promote advantageous immune cell responses in experimental systems for T1D, systemic intravenous delivery of these agents often results in significant harmful off-target effects due to the uncontrolled dosing of bystander cells, tissues and organs. This project focuses on the targeted in vivo delivery of pro-tolerance factors and insulin antigen, in particulate form, targeted to a key immune cell type, dendritic cells (DCs). Dendritic cells, critical for maintenance and initiation of immunity to foregn antigens and tolerance to self-antigens, are phagocytic, antigen presenting cells. This makes DCs an ideal recipient for the targeted delivery of agents provided in particulate form. Moreover, exogenous conditioning of DCs with certain immunomodulatory agents has been shown to induce a pro-tolerance DC phenotype as well as ameliorate T1D. Vaccination with DC-targeting microparticles (MPs) holds promise to correct T1D autoimmune responses, critically, without the costly ex vivo manipulations required of DC-based cellular therapy. This enables the potential for widespread use. Micron-sized biodegradable polymeric particles are phagocytosable, which effectively promotes delivery of encapsulated factors to intracellular sites of DCs over non-phagocytes. These phagocytosable particles can be further targeted to DCs by surface immobilizing molecules targeting DC receptors. Larger (but still small enough to be injectable), non-phagocytosable biodegradable polymeric particles provide controlled release of encapsulated factors to the local extracellular environment at the subcutaneous injection site. Encapsulated factors in these large particles consist of bioactive factors for which DCs have the cognate cell-surface receptors. The objective of this proposal is to engineer a subcutaneously injectable dual MP vaccine system consisting of i.) Phagocytosable DC-targeting MPs delivering antigen and immunomodulatory factor (insulin and vitamin D3) to intracellular sites; and ii.) Non-phagocytosable MPs to deliver, extracellular, factors (GM- CSF and TGF-b1) for DC recruitment and tolerance induction. We expect to effect a pro-tolerogenic DC phenotype and promote induction of regulatory T-cells, suppression of auto-reactive T-cells, and prevent and reverse diabetes in non-obese diabetic (NOD) mice. We hypothesize that the combination of the multiple components in the dual MP system will more effectively provide robust, durable antigen-specific immune suppression than single-component formulations, either in MPs or in soluble form. Aim 1 is to formulate the dual MP system, test it in vitro by characterizing DC phenotype (activated, immature or tolerogenic) and T-cell response (stimulation, Th1, Th2, Treg, or Th17). Aim 2 is to evaluate the ability of the dual MP formulation in vivo, aiming to prevent and reverse diabetes in NOD mice. This novel and innovative approach holds promise for correcting autoimmune responses in T1D and represents a simple, clinically translatable system.